investigating the dosage of the
applied stimulus to pick conditions that

will give meaningful results

1)Lethal conditions may result in data that
are difficult to interpret,

while mild conditions may not
provoke a detectible genomic

expression program

c.Investigate the appropriate
time points for each timecourse experiment

1)Timecourse experiments yield a higher level of detail than
single-

timepoint experiments,
including temporal information

2)Determine the
appropriate time points that will reveal the genomic

expression response … it is easy
to miss rapid responses that occur and subside within a short period (eg
15 minutes)

B.Plan the experiment:ONE VARIABLE ONLY!

As
in any experiment, it is very important to insure only One variable in genomic
expression experiments.Often,
experimental variables that overlooked can provoke substantial changes in
genomic expression and confound analysis of the results.

1.Hypothetical and real examples of
multiple variable experiments:

a.**Diauxic shift during experiment:likely the most common oversight

is the progression of the
cells through diauxic shift, when the cellsbecome limited for glucose and alter their metabolism accordingly.The expression of thousands of genes is
altered during this phase of growth.The timing of diauxic shift is dependent on the culture conditions
(strain, media, growth temperature, aeration, environmental stress) so it is
very important to know when diauxic shift occurs under your conditions and
avoid it (see more below).

b.Pleiotropic drugs
will result in pleiotropic cellular effects and thus genomic

c.Experiments with extensive
cell handling:account for cell
handing in a

control experiment

1)Changes in culture aeration can lead to hypoxia

d.Drugs
suspended in a carrier solution: add carrier alone in mock control

C.Choose the reference for microarrays:The main goal in choosing a reference is to ensure significant hybridization signal in every spot on the
arrays so that the Ratio of R/G signal in each spot can be quantitated …
therefore the identity of the reference is somewhat
arbitrary; the data can be subsequently mathematically
transformed to reveal the biologically-relevant ratios.

1.Example reference
samples

a.Genomic DNA

b.An arbitrary RNA reference pool

c.Time zero RNA, taken just
before beginning the experiment

d.A pool of all of the RNA samples recovered from an experiment

RNA taken from the control sample

2.Regardless of which reference is used, be
sure to use the identical reference

on all arrays in a given timecourse
so as to compare the timepoints to eachother

3.Mathematical transformation example:

a.for a timecourse in which genomic DNA is
used as the reference, there will

be one array
for each time point INCLUDING the time = 0 sample

b. Each ratio for each spot = Red/Green signal =
signal from time point

RNA sample/signal from genomic DNA

c.To transform the data, divide the R/G ratio measured for each gene on
the t>0 arraysby the corresponding
R/G ratio measured on the t = 0array
to cancel the “genomic DNA” denomenator: